NOS3 rs1799983 and rs2070744 polymorphisms and their association with advanced chronic kidney disease and coronary heart disease in Canarian population with type 2 diabetes.

Context: Different polymorphisms of the endothelial nitric oxide synthase gene (NOS3) have been related to diabetic kidney disease. Objective: To evaluate the association between advanced diabetic chronic kidney disease (ACKD) and the rs1799983 and rs2070744 poymorphisms of NOS3 in a population from the Gran Canaria island. Design: Cross-sectional case-control study. Subjects and methods: Polymorphisms were genotyped in 152 subjects with ACKD secondary to type 2 diabetes [estimated glomerular filtration rate (eGFR) <30 mL/min/1.73 m2], 110 subjects with type 2 diabetes for 20 or more years since diagnosis without ACKD (eGFR ≥45 mL/min/1.73m2 and albumin/creatinine ratio <300 mg/g and/or 24-h urinary albumin excretion <300 mg) and 292 healthy controls. Association between both polymorphisms and established coronary heart disease (CHD) was also analyzed in both groups with diabetes. Results: A greater proportion of homozygous individuals for the risk allele C of rs2070744 was found among subjects with ACKD. Association between ACKD and rs2070744 was observed in a recessive genetic model, both for comparison to subjects with diabetes but no ACKD [OR 2.17 (95% CI: 1.17-4.00), p=0.014] and for comparison to healthy controls [OR 1.61 (1.03-2.52), p=0.036]. The frequency of the C allele was significantly higher among subjects with CHD, but only in the group with ACKD. No associations were found for rs1799983. Conclusions: NOS3 rs2070744 is associated with ACKD in population with type 2 diabetes from Gran Canaria. A link between this genetic variant and CHD in Canarian subjects with type 2 diabetes could be restricted to cases with ACKD.

Tyrosinemia type II: Mutation update, 11 novel mutations and description of 5 independent subjects with a novel founder mutation.

BACKGROUND: Tyrosinemia type II, also known as Richner-Hanhart Syndrome, is an extremely rare autosomal recessive disorder, caused by mutations in the gene encoding hepatic cytosolic tyrosine aminotransferase, leading to the accumulation of tyrosine and its metabolites which cause ocular and skin lesions, that may be accompanied by neurological manifestations, mostly intellectual disability. AIMS: To update disease-causing mutations and current clinical knowledge of the disease. MATERIALS AND METHODS: Genetic and clinical information were obtained from a collection of both unreported and previously reported cases. RESULTS: We report 106 families, represented by 143 individuals, carrying a total of 36 genetic variants, 11 of them not previously known to be associated with the disease. Variants include 3 large deletions, 21 non-synonymous and 5 nonsense amino-acid changes, 5 frameshifts and 2 splice variants. We also report 5 patients from Gran Canaria, representing the largest known group of unrelated families sharing the same P406L mutation. CONCLUSIONS: Data analysis did not reveal a genotype-phenotype correlation, but stressed the need of early diagnosis: All patients improved the oculocutaneous lesions after dietary treatment but neurological symptoms prevailed. The discovery of founder mutations in isolated populations, and the benefits of early intervention, should increase diagnostic awareness in newborns.

A novel fibrillin 1 gene mutation leading to marfan syndrome with minimal cardiac features.

Marfan syndrome is an autosomal dominant disorder of the connective tissue, characterized by early development of thoracic aortic aneurysms and/or dissections, accompanied by ocular and/or skeletal involvement, and is caused by mutations in the fibrillin 1 (FBN1) gene. We report on a patient with ectopia lentis and a nonprogressive aortic root dilatation who presented with a novel mutation affecting a conserved cysteine residue present in a calcium-binding epidermal growth factor-like domain of FBN1 (ENSP00000325527, p.Cys538Phe; Chr15:48,805,751 G>T), as revealed by complete sequencing of the FBN1 gene exons and flanking sequences. Identification of the mutation led to genetic screening of apparently asymptomatic family members, allowing the detection of characteristic ocular phenotypes in the absence of typical cardiac Marfan features. This finding stresses the importance of genetic screening of asymptomatic relatives for FBN1 gene mutation carriers.

Novel Frameshift CHD7 Mutation Related to CHARGE Syndrome.

CHARGE syndrome is a rare congenital condition characterized by 6 cardinal features: coloboma, heart defect, atresia choanae, retarded growth and development, genital anomalies, and ear anomalies/deafness. Mutations of the chromodomain helicase DNA-binding protein gene CHD7 are reported to be a major cause of CHARGE syndrome. Herein, we report the case of a 27-year-old patient presenting with typical symptoms who bears a novel heterozygous insertion in exon 2 of the CHD7 gene (c.327dupC) resulting in an amino acid substitution and a frameshift (p.Val110Argfs*22) that leads to a 131-amino-acid truncated polypeptide, likely representing a null allele. Parental genetic screening confirmed the sporadic origin of the mutation.

[Acute renal failure and proximal renal tubular dysfuntion in a patient with acquired immunodeficiency syndrome treated with tenofovir]. / Insuficiencia renal aguda y disfunción tubular proximal en paciente diagnosticado de infección VIH t.

Tenofovir, a new nucleotide reverse transcriptase inhibitor that has good antiviral activity against drug-resistant strains of HIV, is structurally similar to cidofovir and adefovir and seems to be less nephrotoxic. Nephrotoxicity of cidofovir and adefovir is well established and they have been associated with increase for acute renal insufficiency due to tubular toxicity, possibly induced via mitochondrial deplection. Tenofovir has little mithocondrial toxicity in in vitro assays and early clinical studies. However some cases of renal tubular dysfuntion and renal failure related to tenofovir treatment have been published recently. Increased plasma concentrations of didanosine were observed after the adition of tenofovir and protease inhibitors can interact with the renal transport of organic anions leading to proximal tubular intracellular accumulation of tenofovir, yield Fanconi syndrome-type tubulopathy. We present a case in wich acute renal failure and proximal tubular dysfunction developed after therapy with tenofovir in a patiente with HIV who had suffered from complications of didanosine treatment. Although nephrotoxicity certainly occurs much less frequently with tenofovir that it does with other nuclotide analogues, use of tenofovir by patients with underlying renal disfuntion, for longer durations and/or associated with didanosine or lopinavir-ritonavir, might be associated with renal toxicity. Patients receiving tenofovir must be monitored for sings of tubulopathy with simple tests such us glycosuria, phosphaturia, proteinuria, phosphoremia and renal function, as well as assessment for signs of mithocondrial toxicity when a nucleoside analogue is being administered, and therapy should be stopped to avoid the risk of definitive renal failure.

The epithelium-specific ETS protein EHF/ESE-3 is a context-dependent transcriptional repressor downstream of MAPK signaling cascades.

Exon trapping and cDNA selection procedures were used to search for novel genes at human chromosome 11p13, a region previously associated with loss of heterozygosity in epithelial carcinomas. Using these approaches, we found the ESE-2 and ESE-3 genes, coding for ETS domain-containing transcription factors. These genes lie in close proximity to the catalase gene within a approximately 200-kilobase genomic interval. ESE-3 mRNA is widely expressed in human tissues with high epithelial content, and immunohistochemical analysis with a newly generated monoclonal antibody revealed that ESE-3 is a nuclear protein expressed exclusively in differentiated epithelial cells and that it is absent in the epithelial carcinomas tested. In transient transfections, ESE-3 behaves as a repressor of the Ras- or phorbol ester-induced transcriptional activation of a subset of promoters that contain ETS and AP-1 binding sites. ESE-3-mediated repression is sequence- and context-dependent and depends both on the presence of high affinity ESE-3 binding sites in combination with AP-1 cis-elements and the arrangement of these sites within a given promoter. We propose that ESE-3 might be an important determinant in the control of epithelial differentiation, as a modulator of the nuclear response to mitogen-activated protein kinase signaling cascades.

High prevalence of the very rare Wilson disease gene mutation Leu708Pro in the Island of Gran Canaria (Canary Islands, Spain): a genetic and clinical study.

The molecular basis of Wilson disease (WD), an autosomal recessive disorder, is the presence of mutations in the ATP7B gene, a copper transporting ATPase. Hospital records indicated a higher prevalence of WD (1 in 2,600) in some counties in the northeastern region of the island of Gran Canaria (Canary Islands, Spain) that was around 10-fold higher than that described for European populations (1 in 30,000). The ATP7B gene was analyzed for mutations in 24 affected subjects, revealing a high prevalence of the rare Leu708Pro mutation present in 12 homozygous and 7 heterozygous individuals. In these patients, who constitute one of the largest described cohorts of WD homozygotes, we found a variable clinical presentation of the disease, although the biochemical picture was homogenous and characteristic, thereby confirming that the Leu708Pro change is indeed a mutation associated with WD. Haplotype analysis of subjects homozygous for the Leu708Pro mutation showed a conserved shared region smaller than 1 centimorgan (cM), and the region of linkage disequilibrium between the Leu708Pro mutation and neighboring microsatellite markers extended approximately 4.6 cM. When comparing the amount of linkage disequilibrium versus genetic distance from the disease mutation, it was estimated that a common ancestral Leu708Pro chromosome may have been introduced in Gran Canaria over 56 generations ago, dating it back to pre-Hispanic times. The prevalence, and the tight geographical distribution of the Leu708Pro chromosome suggests that the Canary Islands can be considered a genetic isolate for linkage disequilibrium studies.

Analysis of ferrochelatase expression during hematopoietic development of embryonic stem cells.

Ferrochelatase, the last enzyme in the heme pathway, chelates protoporphyrin IX and iron to form heme and is mutated in protoporphyria. The ferrochelatase gene is expressed in all tissues at low levels to provide heme for essential heme-containing proteins and is up-regulated during erythropoiesis for the synthesis of hemoglobin. The human ferrochelatase promoter contains 2 Sp1 cis-elements and GATA and NF-E2 sites, all of which bind their cognate trans-acting factors in vitro. To investigate the role of these elements during erythropoiesis, we introduced expression of the green fluorescent protein (EGFP) transgenes driven by various ferrochelatase promoter fragments into a single locus in mouse embryonic stem cells. EGFP expression was monitored during hematopoietic differentiation in vitro using flow cytometry. We show that a promoter fragment containing the Sp1 sites, the NF-E2 and GATA elements, was sufficient to confer developmental-specific expression of the EGFP transgene, with an expression profile identical to that of the endogenous gene. In this system the -0.275 kb NF-E2 cis-element is required for erythroid-enhanced expression, the GATA cis-element functions as a stage-specific repressor and enhancer, and elements located between -0.375kb and -1.1kb are necessary for optimal levels of expression. Ferrochelatase mRNA increased before the primitive erythroid-cell stage without a concomitant increase in ferrochelatase protein, suggesting the presence of a translational control mechanism. Because of the sensitivity of this system, we were able to assess the effect of an A-to-G polymorphism identified in the promoters of patients with protoporphyria. There was no effect of the G haplotype on transcriptional activity of the -1.1 kb transgene.

Analysis of the human ferrochelatase promoter in transgenic mice.

Ferrochelatase catalyzes the chelation of ferrous iron and protoporphyrin to form heme. It is expressed as a housekeeping gene in all cells, but is upregulated during erythropoiesis. Ferrochelatase activity is deficient in the inherited disease protoporphyria as a result of heterogeneous mutations. Although human ferrochelatase is transcribed from a single promoter in both nonerythroid and erythroid cells, previous studies using transient transfection assays failed to demonstrate erythroid-specific increased expression from 4.0 kb of the human ferrochelatase promoter containing the erythroid cis-elements, GATA and NF-E2. The present study analyzes the in vivo regulation of the ferrochelatase gene to provide insights into the mechanism of its erythroid-specific enhancement. Transgenic (TG) mouse lines were generated in which the luciferase reporter gene was driven by either a 150-bp ferrochelatase minimal promoter (-0.15 TG) or by a 4.0 kb extended 5' upstream region (-4.0 TG). Expression of the -4.0 TG transgene was generally consistent with the endogenous gene during embryonic development and in nonerythroid and erythroid tissues as demonstrated by Northern blotting and mRNA in situ hybridization. The -4.0 TG was expressed at a higher level than the -0.15 TG in nonerythroid and erythroid tissues, including during extramedullary erythropoiesis induced by n-acetylphenylhydrazine injection. The enhanced erythroid expression of the -4.0 TG correlates with the appearance of a DNase I hypersensitive site in the 5' flanking region of the transgene. Therefore, in the context of chromosomal integration, the 5' flanking region of the ferrochelatase gene is necessary and sufficient to confer high levels of transgene expression in erythroid tissue.

A tandem array of Sp-1 sites and a reverse initiator element are both required for synergistic transcriptional activation of the T-cell-specific MAL gene.

We have characterized the three cis elements responsible for promoter strength present in the 5'-flanking proximal region of MAL, a human T-cell-specific gene encoding a proteolipid protein present in detergent-insoluble complexes of high molecular weight. The first element consisted of an initiator sequence that, curiously, was present in reverse orientation compared to that of the standard initiator elements. The other two elements were contained in a region of 126 bp upstream of the mRNA initiation site, and consisted of a tandem array of one GC box and one GA box. The GC box corresponds to a consensus site for the nuclear factor Sp1, whereas the GA box deviates from this consensus, although it was able to compete for the binding of Sp1 in vitro and to respond to trans-activation by Sp1 in vivo. This simple promoter lacks an apparent TATA box and lost more than 99% of its activity when a fragment of 60 bp containing the GC and GA boxes was deleted. A synergistic effect on transcriptional activation was observed in the presence, but not in the absence, of the initiator element when both GC and GA boxes were present.

A single promoter directs both housekeeping and erythroid preferential expression of the human ferrochelatase gene.

We have isolated and characterized the 5'-flanking region of the gene for human ferrochelatase (HFC), the last enzyme of the heme biosynthetic pathway. The proximal promoter of the gene is contained within a region that structurally resembles a CpG island and is devoid of general cis elements such as TATA and CAAT boxes. Recognition sites for the ubiquitous Sp1 family of transcription factors, as well as for the erythroid-specific trans-acting factors NF-E2 and GATA-1 were found, and binding of regulatory proteins to these elements was analyzed by in vitro DNase I protection assays. The contribution of the various cis elements to both ubiquitous and erythroid preferential expression of the HFC gene was assessed by using transient transfection assays. These showed that a minimal Sp1-driven promoter devoid of the upstream erythroid-specific elements was sufficient for erythroid preferential expression of the HFC gene. However, elimination of a repressor sequence lying between the minimal promoter and the erythroid-specific elements resulted in high levels of expression in human erythroleukemic K562 cells only when the cis elements recognized by GATA-1 and NF-E2 were present, suggesting that the activity of these factors is regulated by a downstream repressor in erythroid cells.

Differences in expression of transcription factor AP-1 in human promyelocytic HL-60 cells during differentiation towards macrophages versus granulocytes.

Commitment of HL-60 cells to macrophage or granulocytic differentiation was achieved by incubation with 4 beta-phorbol 12-myristate 13-acetate (PMA) for 30-60 min or with dimethyl sulphoxide (DMSO) for 24 h respectively. The commitment stage towards PMA-induced macrophage differentiation was associated with increases in jun B and c-fos mRNA levels, as well as with an increase in the binding activity of transcription factor AP-1. Nevertheless, gel retardation analysis indicated that the AP-1 activity detected in untreated cells was drastically reduced during the commitment stage of DMSO-induced HL-60 differentiation towards granulocytes. When HL-60 cells were treated with sodium butyrate, which induced monocytic differentiation, a remarkable increase in AP-1 binding activity was detected. Treatment of HL-60 cells with 1 alpha,25-dihydroxyvitamin D3, another monocytic differentiation agent, induced a weak, but appreciable, increase in AP-1 activity. Furthermore, addition of sodium butyrate or 1 alpha,25-dihydroxyvitamin D3 to HL-60 cells induced the expression of c-fos, c-jun, jun B and jun D proto-oncogenes. In contrast, when HL-60 cells were treated with retinoic acid, a granulocytic differentiation inducer, no enhanced AP-1 binding activity was observed, and only a weak increase in jun D mRNA level was detected. These data indicate that formation of AP-1 is not required for the induction of HL-60 differentiation towards granulocytes, whereas induction of monocytic differentiation is correlated with an increase in AP-1 activity. The differential expression of AP-1 activity may be critical in the differentiation of HL-60 cells towards monocytic or granulocytic lineages.

Costimulation of cAMP and protein kinase C pathways inhibits the CD3-dependent T cell activation and leads to a persistent expression of the AP-1 transcription factor.

The effects mediated by a combined stimulation of cAMP- and protein kinase C (PKC)-dependent pathways have been investigated in different cellular systems, and it has been shown that they may complement each other in activating cell proliferation and differentiation. In this report, we show that upon the stimulation of both pathways T lymphocytes became refractory to activation via the CD3/T cell receptor (TcR) complex. T cells preincubated with phorbol 12-myristate 13-acetate (PMA) and dibutyryl cAMP (Bt2cAMP) displayed a deficient proliferative ability in response to anti-CD3 mAb stimulation, whereas lymphocytes treated individually with either Bt2cAMP or PMA responded comparably to untreated samples. We detected an association between the reduced mitogenic response and low expression of both interleukin-2 (IL-2) and the alpha chain (CD25) of the IL-2 receptor (IL-2R). Analysis of intracellular Ca2+ mobilization suggested that the CD3/TcR-dependent signal transduction was impaired in PMA/Bt2cAMP-treated cells. Remarkably, we observed that these samples displayed a persistent expression of the c-fos protooncogene, associated to an increased AP-1 DNA-binding activity, whereas no variations of CREB or NF-kB were detected. Neither Bt2cAMP nor PMA individually mediated these sustained effects, which therefore appear as a consequence of the interplay between both metabolic stimuli. Altogether, the data provide the evidence that both pathways complement each other in regulating gene expression and, conversely, downregulate the TcR transduction mechanisms.

Activation of activating protein 1 during hepatic acute phase response.

During an acute phase response following inflammatory stimuli, specific changes occur in the synthesis and secretion of many hepatic proteins. Because the expression of differentiated function requires the coordinated regulation of many genes, we investigated the activity of general and tissue-specific transcription factors using a rat liver model of the acute phase response induced by Freund's adjuvant. Nuclear extracts and RNAs were prepared throughout a 48-h posttreatment period. Mobility shift assays revealed increased binding activity by nuclear factor-kappa B, interleukin-6 (IL-6) responsive element binding protein, and activating protein 1 (AP-1). Two AP-1 complexes were induced during the acute phase response, and correlation between their presence and transcription activity was demonstrated by transfection studies. Elevated binding activity of AP-1 also correlated with elevated levels of c-jun, junD, junB, and c-fos mRNAs. Western blots showed elevated hepatic levels of c-Jun but not c-Fos proteins during the acute phase response. In addition, IL-6, tumor necrosis factor-alpha, and IL-1 beta, cytokine regulators of the acute phase response, stimulated expression of an AP-1 responsive reporter gene introduced by DNA-mediated transfection into adult rat hepatocytes in primary culture. These findings demonstrate the complexity of AP-1 hepatic transcription factor responses to humoral regulators with direct hepatocellular effects.

Characterization of the p150,95 leukocyte integrin alpha subunit (CD11c) gene promoter. Identification of cis-acting elements.

The leukocyte integrin p150,95 (CD11c/CD18) is involved in a number of cell-cell and cell-extracellular matrix interactions and mediates signal transduction into the cytoplasm. p150,95 is expressed on cells of the myeloid lineage as well as on certain activated T and B lymphocytes, and its expression is regulated during cell activation and differentiation. Since CD18 is expressed on all leukocyte lineages, the restricted expression of p150,95 must be controlled at the level of CD11c gene transcription. To understand the mechanisms that direct the constitutive and regulated leukocyte expression of p150,95 we have structurally characterized the CD11c promoter region and initiated its functional dissection. The CD11c promoter lacks TATA- and CCAAT-boxes, directs the synthesis of transcripts with heterogeneous 5'-ends, and contains an initiator-like sequence at the major transcription initiation site. Several putative binding sequences for ubiquitous (Sp1, AP-1, AP-2, and NF-kB) and leukocyte-specific (PU.1) transcription factors have been identified in the proximal region of the CD11c promoter which may participate in the regulation of the expression of p150,95. Transient expression of CD11c-based reporter gene constructs indicates that the CD11c promoter dictates the tissue-specific expression of p150,95 and that sequences contained within 160 base pairs 5' from the major transcriptional start site are involved in the tissue-specific and regulated expression of p150,95. DNase I protection analysis on the promoter region spanning from -160 to +40 revealed four regions of DNA-protein interactions (FPI-FPIV), two of which (FPII and FPIV) correlate with the cell type-specific and regulated expression of the CD11c gene.